Method for the separation of oligomeric n-substituted (meth)acrylamide compounds and conjugates thereof which are reversibly thermally precipitating

ABSTRACT

The invention relates to a method for the separation of oligomeric N-substituted (meth)acrylamides and conjugates thereof (enzyme conjugates, affinity macroligands and AML complexes) which are reversibly thermally precipitating from aqueous solutions, whereby the oligomeric N-substituted (meth)acrylamides and conjugates thereof are precipitated thermally and filtered in the presence of salts and filtration adjuncts. The oligomeric N-substituted (meth)acrylamides and conjugates thereof are characterised by a separation rate of over 90% of the thermal precipitate and thus permit an efficient biocatalysis and affinity preparation based on oligomeric compounds which are reversibly thermally precipitating on a large scale.

[0001] The present invention relates to a method for separatingreversibly thermally precipitatable oligomeric N-substituted(meth)acrylamides and their conjugates from aqueous solution, as well asto separated, thermally precipitatable oligomeric N-substituted(meth)acrylamides and their conjugates, synthesized by the method.

[0002] In the area of polymer synthesis, the free radical telomerizingover a chain transfer reagent is a customary method of synthesizing alinear, low molecular weight polymer (having a degree of polymerizationof less than 100 and molecular weights distributed very homogeneously)of controlled chain length and with a terminal functional group.Covalently bound conjugates, such as enzyme conjugates and affinitymacroligands (AML), can be synthesized over the functional group fromthe oligomers so produced.

[0003] A plurality of N-substituted (meth)acrylamides form water-solublepolymeric compounds, which precipitate reversibly from water above alower critical demixing temperature, called the LCST (lower criticalsolution temperature). A list of monomers, which come intoconsideration, is given in the U.S. Pat. No. 5,162,582. The LCST isfixed by varying the monomer chemistry or the copolymer composition. Itis, for example, 32° to 34° C. for poly-N-isopropylacrylamide in water.The LCST is independent of the chain length of the polymers and of thepH of the solution; as a rule, it is lowered by salts as a function oftheir molarity.

[0004] In methods known from the art, homogeneous, reversibly thermallyprecipitatable oligomers with terminal, functional groups aresynthesized by telomerization in organic solvents or water. The yield isbetter than 60%. These oligomeric compounds are purified by repeatedsoluble-insoluble precipitations in organic solvents, such as acetone inhexane, with subsequent filtration and vacuum drying. The repeatedpurification is necessary in order to free the oligomeric preparationfrom the toxic monomers.

[0005] However, this purification method requires relatively largevolumes of organic solvents having low water content. Experience hasshown that, for a working-up step, approximately one liter of n-hexanewith a water content of less than 0.05 percent is required in order toprecipitate 10 g of oligomeric compound. The low water content of theorganic solvent is required in order to avoid gelatinization of theoligomers during the precipitation and, with that, keep the oligomericaggregates in a filtratable form. Accordingly, aside from the high costsfor solvents and the use of many personnel for this purification method,which can be automated only with difficulty, this method is also verydisadvantageous for reasons of operational safety and environmentalprotection, when used on an industrial scale with the volumes ofsolvents required for such a purpose.

[0006] The reversibly thermally precipitatable oligomers, the so-called“smart polymers” have a diversified area of applications inbiotechnology, for example, in biocatalysis and bioseparation (affinityprecipitation). For example, J. -P. Chen (J. Chem. Technol. Biotechnol.73 (1998) 137-143) describes a conjugate with α-chymotrypsin, the enzymeactivity and thermal stability of which are greater than those of nativeenzyme. For biocatalysis, such enzyme conjugates offer the advantagesthat the catalysis precedes homogeneously and that the biocatalysts caneasily be separated by thermal precipitation and used once again.

[0007] Affinity precipitation is a bioseparation method, which utilizesthe precipitation properties of the oligomers in combination withligands, which have a specific affinity to a target substance, in orderto separate and purify this target substance specifically.

[0008] In the WO 01/25287 A1, AML are described, which can be usedefficiently for the purification of proteins and nucleic acids. However,the oligomers were produced only in small amounts and where purifiedeither by precipitation in organic solvents or by diafiltration followedlyophilization. The latter method, however, is very time-consuming andcan be used only for relatively small amounts. For the separation ofthermally precipitatable oligomers and AML (including AML targetsubstance complexes), centrifugation was selected because only smallvolumes were biopurified.

[0009] However, the industrial application of “smart polymers” inbiocatalysis and bioseparation requires, on the one hand, a method forthe synthesis of monomer-free oligomers on a large scale and, on theother, a method for the efficient separation of the thermal precipitatesfrom a large volume. However, it is a common feature of all previouslydescribed examples of the separation of reversibly thermallyprecipitatable oligomers and their conjugates (enzyme conjugates, AMLand AML target substances) from aqueous solution, that the precipitateseparation was carried out by centrifugation using only using smallvolumes.

[0010] Y. G. Takei et al. (Bioconjugate Chem. 4 (1993) 42-46) have shownthat, especially in the case of dilute solutions, low molecular weightoligomers require very high centrifugal accelerations, in order toseparate more than 80 percent of the thermal precipitate. In the case ofa 1 percent by weight solution of an oligomer with an average molecularweight of about 2,500 g/mole, only about 60 percent can be separated asprecipitate even an a centrifugal acceleration of 10,000 g. Moreover,centrifugation has the disadvantage that it produces very compactprecipitate gels, which can be dissolved again only very slowly.Moreover, it can be used only in batch operation and, with that, forrelatively small volumes, since the thermal precipitate, because of itsgelatinous consistency, cannot be supplied continuously.

[0011] Y. G. Takei et al. (Bioconjugate Chem. 4 (1993 42-46) have alsoshown that, in the case of oligomers with an average molecular weight ofless than 5,000 g/mole, the filtration of the precipitate causes highlosses. For example, only about 20 percent of precipitated oligomerswith an average molecular weight of about 2,500 g/mole can be separatedby filtration from a 1 percent by weight solution; at 10 percent byweight, the recovery rate increases to about 70 percent.

[0012] Accordingly, neither methods, which enable reversibly thermallyprecipitatable oligomers to be purified in large amounts without usingorganic solvents, nor methods, with which reversibly thermallyprecipitatable oligomers and their conjugates can be separatedefficiently from aqueous solutions of large volume, are known from thestate of the art.

[0013] It is an object of the present invention to make available aworking up and separating method, which enables reversibly thermallyprecipitatable oligomers and their conjugates to be separatedefficiently from an aqueous solution.

[0014] It is a further object of the present invention to make availableoligomeric N-substituted (meth)acrylamides with an improved separationrate.

[0015] Pursuant to the invention, the objective is accomplished owing tothe fact that the oligomeric N-substituted (meth)acrylamides and theirconjugates are thermally precipitated and filtered in the presence ofsalts and in the presence of filter aids.

[0016] It was determined that oligomers and their conjugates can beseparated rapidly and efficiently by the method of the present inventionfrom any volume, for example, from 1 ml to 10, 000 L of aqueoussolution. With that, monomer-free (containing less than 1 ppm ofmonomer) oligomeric N-substituted (meth)acrylamides can be prepared byrepeated separations or washings.

[0017] It is particularly advantageous that, with the method of thepresent invention, oligomers with an average molecular weight between500 g/mole and 5,000 g/mole can be separated effectively. This is ofparticular advantage since, only in the case of short-chain oligomers,is the full coupling activity of the terminal, functional groupsrealized. In addition, only in the case of enzyme conjugates and AMLwith such short-chain oligomeric compounds, is a high enzyme activityand a diffusion-free and homogeneous affinity interaction assured.

[0018] It is equally advantageous that, with the method of the presentinvention, N-substituted (meth)acrylamide conjugates can be separatedeffectively from dilute solutions. It is a distinguishing feature ofbiocatalysis and biotechnological purification methods that thecatalysts and the target substances, which are to be purified, are usedor present only in small concentrations. For this reason, a certainamount of oligomer must be added as precipitation promoter to thereversibly thermally precipitatable oligomeric conjugates in order toachieve a good degree of separation. The amount of promoter must bematched to the desired degree of separation and degree of activity ofthe biocatalysts or AML. The degree of non-specific adsorption and/orabsorption must also be taken into consideration when adding to thepromoter. The addition of promoter should therefore be kept to aminimum. It has proven to be advisable that the total amount ofoligomeric compounds and of their conjugates in aqueous solution isbetween 0.1 percent by weight and 10 percent by weight and preferablybetween 0.8 percent by weight and 3 percent by weight.

[0019] Appropriately, the oligomers and their conjugates have an LCST inwater of between 10° C. and 80° C. At an LCST about 80° C., it istechnically difficult to carry out a thermal precipitation. At an LCSTbelow 10° C., it may become impossible to dissolve the precipitate whensubstances, which lowered the LCST, are added. Withpoly-N-isopropylacrylamide, poly-N-n-propylacrylamide,poly-N-acryloylpyrolidine and the co-oligomers, composed ofN-isopropylacrylamide and N,N-dimethylacrylamide in a molar ratio of80:20, said LCST range is covered. The corresponding monomers arechemically simple and can be produced economically.

[0020] It is furthermore advantageous if the thermal precipitation iscarried out in the presence of salts. At a temperature above the LCST,pure oligomers form finely divided, unstable aggregates, which can beseparated only to an inadequate degree. Aside from the, as a rule,LCST-lowering effects, salts, at low molarities, have a decisive effecton the aggregation behavior of thermal precipitates. For example, in thepresence of sodium acetate, very fine, separate precipitate particlesare formed, which have a tendency to float. On the other hand,gelatinous, coherent precipitates frequently are formed with chloridesalts. The selection of salts, their mixture and amount can only be madeempirically for the special application.

[0021] In this connection, the aggregation-promoting properties as wellas the LCST-lowering properties of the salts must be taken intoconsideration. A total salt concentration of between 0.01 M and 3 M isadvantageous. For most applications, however, total sold concentrationsof 0.5 M to 1.5 M are sufficient to affect the aggregation-behavior ofthe precipitate, so that a high separation rate can be achieved whilefiltering.

[0022] Furthermore, the temperature above the LCST, at which theoligomers and their conjugates are precipitated and filtered, isimportant. Advisably, the precipitation temperature and also thefiltration temperature is 1° C. to 20° C. above the LCST. At atemperature more than 20° C. above the LCST, losses occur, because theprecipitate aggregates dissolve once again. A temperature range of 5° C.to 10° C. above the LCST is preferred. It is important that thetemperature does not to drop during the filtration. If it were to drop,there could also be losses during the separation because the precipitatedissolves once again.

[0023] Filter aids are of decisive importance for separating thermallyprecipitatable N-substituted (meth)acrylamides and their conjugatesefficiently from aqueous solution. It has proven to be appropriate touse a cellulose with the following, additional properties as filter aid:

[0024] proportion of cellulose: ≧99 percent

[0025] wet density: 0.2-0.3 g/cc

[0026] fiber length: <75 μm (99%), <32 μm (65%).

[0027] This has the advantage that thermally precipitatable oligomericcompounds can be separated efficiently with a degree of separation ofmore than 90 percent, an adequately high filtration flow and a moderatedifferential pressure. The filter aids may be added already during thethermal precipitation or to the oligomeric compounds, which have alreadybeen precipitated thermally. The separation of a thermal precipitateover a bed of filter aid is also possible. The most advantageousvariation is the presence of filter aids during the precipitation. Bythese means, the filter aid is incorporated in the aggregating thermalprecipitate and the separation of the finest aggregates accordinglybecomes possible. It is furthermore advantageous that, especially whencellulose is used, the precipitates can rapidly be dissolved once againat a temperature below the LCST. Aside from the efficiency, the use of afilter aid of high purity has the advantage that its surfaces arestandardized and, with that, their properties can be controlled and/ormodified. Above all, for affinity precipitation methods, losses due toadsorption at the surface of the filter aid and/or non-specificadsorption effects can be minimized.

[0028] However, the amount of filter aid, based on the weight percent ofthe precipitate from the solution, which is to be filtered, thefiltration area and the magnitude of the maximum filtration pressure to,which is to be applied, can only be determined empirically.

[0029] The invention is explained in greater detail by means of anexample.

EXAMPLE 1

[0030] Measurement Methods:

[0031] ICP Atom Emission Spectroscopy

[0032] The amount of oligomer in aqueous solution was determined fromthe sulfur content by means of ICP-AES (Plasma 1000 of Perkin Elmer) ata wavelength of 182.037 nm with automatic background correction andcalibration with a sulfuric acid solution.

[0033] Reverse Phase HPLC

[0034] The N-isopropylacrylamido monomer concentration was determined bymeans of a reverse-phase Zorbax Rx C18 column (4.6 mm×25 cm) ofHewlett-Packard and an HPLC system, consisting of a TechLab binary pump,the ERC-3112 solvent degasser of ERMA CR and the UV/Vis SPD-10A detectorof Shimadzu under the following conditions: Solvent A water Solvent Bacetonitrile 0-98% gradient in 30 minutes Flow rate 1 mL/min. Injectionvolume 20 μL Detection UV @ 214 nm

[0035] Oligomeric N-isopropylacrylamide (M_(n)=2,300 g/mole) wassynthesized by telomerization form N-isopropylacrylamide andazoisobutylnitrile in methanol at 65° C. Subsequently methanol wasdistilled off under vacuum and the oligomer was taken up in water.

[0036] To 150 ml of the aqueous oligomeric solution, cellulose(Diacel-75, trademark of the CFF Co.) and 8.766 g of sodium chloridewere added at 15° C. (LCST=22° C.). The solution was heated withstirring to 30° C. and filtered in a thermostated filter funnel (10 cc:filter area, 200 ml capacity) over a PVDF warp/PTFE filling monofilament(11.5 μm pore size) as filter medium. Without pressure, a clear filtratewith an average flow of more than 4 m³/m²h and a temperature of 29° C.was obtained. The filter cake was resuspended in 1 M sodium chloride at15° C., and the oligomer was separated once again by thermalprecipitation. This procedure was repeated a total of three times.

[0037] ICP-AES measurements of filtrate samples showed that, incomparison to standard measurements, more than 97 percent of theoligomers were separated from a 0.5 percent by weight solution duringthe last separation process. HPLC measurements of the filter cake showthat the monomer contamination was reduced by a factor of 44 in onefiltration step in comparison to standard measurements.

1. A method for the separation of reversibly thermally precipitatableoligomeric N-substituted (meth)acrylamides and their conjugates fromaqueous solution, wherein the oligomeric N-substituted (meth)acrylamidesand data conjugates are precipitated thermally and filtered in thepresence of salts and in the presence of filter aids.
 2. The method ofclaim 1, wherein the oligomers consist of repeating units ofN-isopropylacrylamide, N-n-propylacrylamide or N-acryloylpyrrolidine orof co-oligomers of N-isopropylacrylamide and N,N-dimethylacrylamide in amonomeric ratio of 80:20.
 3. The method of claims 1 and 2, wherein, assalts, potassium, sodium, ammonium, calcium and/or magnesium salts ofchloride, acetate and/or sulfate, as well as their mixtures are used. 4.The method of claim 3, wherein the total concentration of the salts isbetween 0.01 M and 3 M.
 5. The method of claims 1 to 4, wherein thetotal amount of the oligomeric N-substituted (meth)acrylamides and theirconjugates in aqueous solution is between 0.1 percent by weight and 10percent by weight.
 6. The method of claims 1 to 5, wherein theoligomeric N-substituted (meth)acrylamides and their conjugates areprecipitated thermally at 1° C. to 20° C. and preferably at 5° C. to 10°C. above the LCST and filtered at the same temperature.
 7. The method ofclaims 1 to 6, wherein celluloses with the following properties are usedas filter aids, proportion of cellulose: ≧99 percent wet density:0.2-0.3 g/cc fiber length: <75 μm (99%), <32 μm (65%).
 8. A mixture offilter aids and reversibly thermally precipitatable oligomericN-substituted (meth)acrylamides and their conjugates, separated by themethod of claims 1 to 7, wherein the separation rate of the filter aidand the reversibly thermally precipitatable, oligomeric, N-substituted(meth)acrylamides and their conjugates is more than 90 percent.
 9. TheN-substituted (meth)acrylamide compounds of claim 8, wherein thereversibly thermally precipitatable N-substituted (meth)acrylamides havean average molecular weight between 500 g/mole and 5,000 g/mole.
 10. TheN-substituted (meth)acrylamide compounds of claim 8, wherein the LCST inwater of the reversibly thermally precipitatable N-substituted(meth)acrylamides and their conjugates is between 10° C. and 80° C.